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Title: Structural and compositional studies of the type III secretion system of shigella flexneri
Author: Cheung, Martin Philip
Awarding Body: University of Bristol
Current Institution: University of Bristol
Date of Award: 2013
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Shigella flexneri is the causative agent of bacillary dysentery and utilises the Type III Secretion System (T3SS) for cellular invasion. The central component of the T3SS is the needle complex(NC), which resembles a molecular 'syringe' and is used by S. flexneri to secrete proteins directly into the host cell, thereby instigating the invasive process. After host cell sensing and activation via the tip complex (TC), which sits at the extremity of a 50 nm-Iong needle, a signal is thought to be transmitted from the needle to the base, resulting in activation of protein secretion. Precisely how host cell sensing occurs and how this leads to protein secretion in the base remains to be fully elucidated. Here, we used electron microscopy (EM) and mass spectrometry (MS) to investigate the structure of the NC with a view to decipher the structural basis of its function. Utilising cryoEM, we solved the structure of the needle in both wild type and activated states to sub-nanometer resolution, allowing us to visualise key inter-subunit interactions made by the needle subunit, MxiH. Whilst no structural differences were seen between wild type and activated needles, a previously unidentified hairpin motif was elucidated, which was shown to form part of a crucial inter-subunit junction required for needle polymerisation and possibly signal transduction. Using negative stain EM, we produced a moderate resolution density map of the wild type TC, revealing four copies of protein IpaD and one copy of IpaB. The reconstruction showed the subunits to be tilted, resulting in a proposed metastable TC. Reconstructions of TC's in partially activated and fully activated states revealed a correlation between subunit tilting/bending/rotating and activation, allow us to propose a mechanism for TC function. Furthermore, we show that the TC subunits probably interact with the needle via their C termini and with the crucial needle junction. In order to elucidate the architecture of the base, we used EM and Absolute Quantification (AQUA) peptides to show that the NC outer membrane ring(OMR) has C IS rotationally symmetry and that the inner membrane ring (IMR) has 24 subunits, possibly arranged in a C 12 configuration. We also show that each NC possibly has one copy of the export apparatus protein MxiA. We also report here the transmission of a 3.9MDa complex by native MS, the largest membrane protein analysed by this method and possibly representing the mass of the full intact Ne. Finally, we detail a new method for cryoEM grid preparation that overcomes a long standing problem in cryoEM of macromolecular complexes - sample adsorption to the grid surface - which has the potential to be applied to a diverse range of samples.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available